Multiplex polymerase chain reaction (PCR) with color-tagged module-shuffling primers for comparing gene expression levels in various cells

C. Uematsu, J. Nishida, K. Okano, Fumihito Miura, Takashi Ito, Y. Sakaki, H. Kambara

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

A method based on the multiplex polymerase chain reaction (PCR) and gel electrophoresis for the comparative analysis of gene expression levels was developed. Using the method many cDNA fragments from different sources can be compared simultaneously. Competitive PCR amplification of expressed genes from different sources was performed by using 'module-shuffling primers' (MPSs). The MPSs (labeled with different fluorophores) consist of sequence modules of 3 or 4 nt. The modules are arranged in different orders in each primer; therefore, the base sequences of the primers are different but their melting temperatures are identical. The genes expressed in different sources are ligated with tags complementary with the MPSs. Tag-ligated fragments are mixed in one tube and amplified at the same amplification efficiency by the MPSs. Amplified fragments are detected separately by multiple-color gel electrophoresis. This method can detect different amounts of each expressed gene, up to a difference in amounts of 30%, and its detection limit is 0.1 amol per assay.

Original languageEnglish
Number of pages1
JournalNucleic acids research
Volume29
Issue number16
Publication statusPublished - Aug 15 2001
Externally publishedYes

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Multiplex Polymerase Chain Reaction
Color
Gene Expression
Electrophoresis
Gels
Gene Amplification
Freezing
Genes
Limit of Detection
Complementary DNA
Polymerase Chain Reaction
Temperature

All Science Journal Classification (ASJC) codes

  • Genetics

Cite this

Multiplex polymerase chain reaction (PCR) with color-tagged module-shuffling primers for comparing gene expression levels in various cells. / Uematsu, C.; Nishida, J.; Okano, K.; Miura, Fumihito; Ito, Takashi; Sakaki, Y.; Kambara, H.

In: Nucleic acids research, Vol. 29, No. 16, 15.08.2001.

Research output: Contribution to journalArticle

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